Project Summary/Abstract ! Most genetic recorders described so far utilize the CRISPR/Cas9 system to leave a unique genetic scar in each cell that can be traced in daughter cells. However, since these systems are largely deletion based, there is a finite number of events and lineages that they can be used to trace. Hence all these recorders are fundamentally restricted in the number of lineages they can trace since development in mammals is a prolonged process, with radial glia in the brain dividing greater than 50 times. This central limitation precludes the utility of these approaches in mammalian model organisms that are most relevant to human development. Moreover, these technologies do not allow dynamic recording of biological events throughout the life-cycle of an animal. Thus, a different approach to lineage-tracing and biological recording is needed that does not reach saturation and will permit chronicling lineages in whole organisms and complex tissues. Here we propose to exploit recently described base-editors to enable continuous, dynamic, genetic recording in non-human primates (NHPs), such as a marmoset. Our strategy, called CHRONICLE, bypasses many of the limitations of currently used systems. CHRONICLE (Cellular Hierarchy Recording in Organisms by Nucleotide Interconversion with Cas9 Linked Editors) combines base-editing with self-targeting guide RNA arrays to generate a large repertoire of sequence variants that can be used to trace cellular hierarchy lineages in complex mammalian brains. This tool combined with single cell RNA sequencing (scRNA) will enable us to probe the developmental differences between the human and marmoset cortex and the lineage trajectory of cell types found in each species and the circuits they might contribute to. In Aim 1, we propose to use CHRONICLE to trace lineages in vitro in cortical organoids derived from human embryonic stem cells (hESCs) to trace the intrinsic properties of neural progenitors in a controlled environment. In Aim 2, we will introduce CHRONICLE into marmoset cortical organoids, derived from marmoset induced pluripotent stem cells (iPSCs), as well as marmoset pre-neurulation embryos. This will allow a comparison between in vitro and in vivo development. Finally, in Aim 3, we will use the data obtained from Aims 1 and 2 to compare developmental lineage tree in the marmoset cortex by phylogenetic reconstruction of CHRONICLE evolution in projection neurons, interneurons, and glia using scRNA analysis. This approach will permit chronicling lineages dynamically in a primate brain and bypass many of the limitations of current CRISPR-based molecular recorders. Since many stages of cortical development are unique to primates, this study has the potential to reveal novel lineage relationships and developmental milestones for the first time, and provide answers to key developmental questions. Solving the lineage tree of a primate brain will impact our ability to utilize developmental principles to restore function to diseased and damaged tissues.